Non-typhoidal Salmonella (NTS) are increasingly being recognized as important causes of invasive disease (e.g. sepsis, meningitis) in the very young and the elderly in the USA. The growing resistance of NTS strains to multiple antibiotics further complicates treatment. NTS disease in the USA is accounted for primarily by serovars belonging to three serogroups (B, C and D). Our overall goal is to develop a broad-spectrum vaccine against these invasive NTS serogroups. We have already developed live oral and conjugate vaccines against Group B {Salmonella enterica subspecies enterica serovar Typhimurium) and D (S. Enteritidis) serovars that can protect against invasive NTS disease with the wild-type homologous pathogen. Although less common than strains of Salmonella Groups B and D, Group C organisms represent a significant proportion of NTS cases, and some Group C serovars (e.g. S. Choleraesuis) are highly invasive. The purpose of this translational research proposal is to develop Salmonella Group C live attenuated and conjugate vaccines. Our central hypothesis is that appropriately engineered attenuated strains of Salmonella enterica Group Cl and C2 serovars can: 1) allow safe, high yield preparation of core-0 polysaccharide (COPS) and flagella protein for making conjugate vaccines, and 2) serve as protective live attenuated vaccines. We will select suitable Salmonella Group Cl and C2 strains and genetically engineer them so that they are attenuated and secrete large amounts of Phase 1 flagellin protein into the supernatant. We will purify COPS and flagellin from these strains and construct COPS-FliC conjugate vaccines using various chemical strategies. These conjugates will be evaluated in adult and aged mice. Live attenuated Salmonella Group Cl and C2 vaccine strains will also be evaluated for their ability to protect adult and aged mice. We will also determine whether gnotobiotic piglets can be protected from invasive disease by S. Choleraesuis conjugate and live oral vaccines. We will ascertain whether NTS vaccines can also protect against gastroenteritis by testing our live attenuated S. Typhimurium vaccine CVD 1931 in the rhesus macaque model of Salmonella gastroenteritis. Finally, we will determine whether a multivalent formulation of live oral or conjugate Group B, C and D Salmonella vaccines can prevent invasive disease caused by Group B, C and D serovars. If we are successful, these results will pave the way for initiating future Phase 1 clinical trials and we will have addressed three ofthe four broad objectives of this multi-center research proposal.